More than two million new cases of cancer are reported annually in the seven major worldwide pharmaceutical marketplaces (US, Japan, Germany, Italy, France, Spain, UK) (Krul, 1994). Chemotherapy is an important part of modern clinical cancer treatment for human malignancies. However, chemotherapy frequently is ineffective due to either endogenous or acquired tumor cell resistance. Typically, the resistance is developed simultaneously to a wide range of structurally unrelated chemotherapeutic drugs with different mechanisms of action and therefore is called multidrug resistance (MDR) (Deuchards and Ling, 1989; Pastan and Gottesman, 1987). Generally, only 5-10% of new cancer cases will respond successfully to chemotherapy, and 40-45% of cancer patients will annually develop MDR to their particular chemotherapeutic regimens.
Several mechanisms can account for MDR at the molecular and cellular level. Decreased drug uptake or increased drug efflux, altered redox potential, enhanced DNA repair, increased drug sequestration mechanisms or amplification of the drug-target protein all are postulated cellular mechanisms for expression of tumor cell drug resistance to various chemotherapeutic agents. One of the most thoroughly studied mechanisms by which tumor cells acquire MDR is overexpression of a transmembrane glycoprotein, called P-glycoprotein (Pgp). Pgp is thought to act by rapidly pumping hydrophobic chemotherapeutic agents out of tumor cells, thereby decreasing intracellular accumulation of certain chemotherapeutic agents below their cytostatic concentrations. Various compounds have been identified, such as tamoxifen, cyclosporin A, and SDZ PSC 833, that are able to reverse MDR. These agents, termed MDR modulators, while not chemotherapeutic drugs themselves, are important in enhancing the cytotoxicity of chemotherapeutic agents by restoring sensitivity in an otherwise resistant setting (Fan, 1994).
A major challenge in cancer chemotherapy is to understand the molecular mechanisms by which MDR modulators reverse drug resistance. The action of MDR modulators is dependent in part on interaction with the biochemical and physiological processes that evoke the resistance phenomenon. It has been shown that some MDR modulators bind directly to Pgp (Yusa, 1989; Foxwell, 1989) and thereby interfere with binding and export of anticancer agents to the drug pump. Tamoxifen, an antiestrogen compound used in treatment of breast cancer long known for MDR modulatory properties (Fan, 1994), binds to Pgp (Callaghan and Higgins, 1995), as does the nonimmunosuppressive cyclosporine A analog, PSC 833 (Archinal-Mattheis, 1995), a potent drug resistance modulator (Gaveriaux, 1995). PSC 833 has been shown to be significantly more effective than verapamil and cyclosporine A in reversing MDR, in vitro and in vivo (Watanabe, 1995). However, it has been observed that PSC 833, unlike cyclosporine A, is a strong agonist in glycolipid metabolism and elicits ceramide formation whereas cyclosporine A does not (Lucci, 1997).
Recently, it has been suggested that the development of MDR is closely related to a unique glycosphingolipid pattern within the cancer cell. It has been shown that MDR cells, as opposed to drug-sensitive cells, display increased levels of glucosylceramide (Lavie, 1996). Subsequent findings indicate that MDR modulators may increase the cellular susceptibility to chemotherapeutic agents through regulation of ceramide metabolism in cancer cells (Lavie, 1997).
Ceramide is a well-known second messenger, stimulating specific kinases, phosphatases, and transcription factors that mediate a variety of cellular functions (Hannun, 1994; Hannun and Obeid, 1995; Jarvis, 1996). It is the backbone of all sphingolipids, including sphingomyelin, and glycosphingolipids and, thus, is subject to complex metabolic regulation. Ceramide has been reported to initiate differentiation and cell proliferation, and also is known to serve as a second messenger for apoptosis (Obeid, 1993; Pena, 1997). Ceramide is reported to be the messenger of signaling events that originate from different cell surface receptors, including interferon-γ, TNF-α, interferon-1β, CD95 (Fas/APO-1), nerve growth factor receptor, and CD28 (Testi, 1996; Symth, 1997; Haimovitz-Friedman, 1997). Ceramide also appears to be involved in the action of PKC ζ, Vav protooncogene, 1α-25-dihydroxy vitamin D3, dexamethasone, ionizing radiation, and chemotherapeutic agents (Testi, 1996; Symth, 1997; Haimovitz-Friedman, 1997). There is also data suggesting that loss of ceramide production is one cause of cellular resistance to apoptosis induced by either ionizing radiation, TNF-α, or adriamycin (Chuma, 1997; Cai, 1997; Michael, 1997; Bose, 1995; Santana, 1996; Zyad, 1994; Lavie, 1997; Cabot, 1997).
Ceramide is produced by either (i) condensation of the sphingoid base sphinganine and fatty acyl-CoA by the enzyme ceramide synthase and subsequent oxidation, (ii) by degradation of sphingomyelin into phosphorylcholine and ceramide by the action of sphingomyelin-specific forms of phospholipase C, or (iii) by degradation of glucosylceramide by β-glucosidase (glucocerebrosidase). The ceramide formed then is metabolized to sphingomyelin or glucosylceramide by addition of the appropriate head group.
Glucosylceramides are the most widely distributed glycosphingolipids in cells. Glucosylceramides are produced by glucosylceramide synthase (GCS) transferring glucose from UDP-glucose to ceramide (Basu, 1968). Recently, it has been shown that human GCS is a glycoprotein containing 394 amino acids encoded from 1,182 nucleotides including a G+C-rich 5′ untranslated region of 290 nucleotides (Ichikawa, 1996). Glucosylceramides serve as precursors for the biosynthesis of over 200 known glycosphingolipids. In addition to their role as building blocks of biological membranes, glycosphingolipids have long attracted attention because of their putative involvement in cell proliferation (Hannun and Bell, 1989), differentiation (Schwarz, 1995; Harel and Futerman, 1993), oncogenic transformation (Hakomori, 1981; Morton, 1994) as well as their role in escape from onset of apoptosis (Nakamura, 1996).
Apoptosis or programmed cell death is widely recognized to be a cellular mechanism crucial for toxic response to chemotherapeutic agents (Wyllie, 1997). This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Recent studies have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals including hormones, serum growth factor deprivation, chemotherapeutic agents, and ionizing radiation. A substantial body of evidence now exists defining ceramide as a messenger for the induction of apoptosis. In intact cells, rapid ceramide generation is an early event in the apoptotic response to numerous stimuli including cytokines and environmental stresses, and ceramide analogs mimic the effect of stress and induce apoptosis (Hannun, 1994; Kolesnick and Golde, 1994; Hannun and Obeid, 1995; Jarvis, 1996).
It is apparent that agents which affect ceramide metabolism and thus apoptosis have tremendous therapeutic utility for a wide variety of diseases, such as cancer, where regulation of apoptosis and proliferative capacity of tumors are tightly coupled. However, it is particularly difficult to screen for agents based on their apoptotic modulating activity, since such assays require a cell line that can be maintained in vitro and retain sensitivity to apoptosis modulating signals. Additionally, apoptosis endpoint screening assays are cumbersome, time consuming, and at best not reliable due to variability of results.
Various methods exist to test potential therapeutic agents in both the preclinical setting and in clinical trials of individualized patient-specific therapies. These include, among others, the human tumor cloning assay (Shoemaker, 1985), dye exclusion assays (Weisenthal, 1983), adhesive tumor cell culture systems (Ajani, 1987), and multicellular tumor spheroids (Yuhas, 1978). However, most of these are labor intensive when testing for drug sensitivities against single agents, and even more unwieldy when used in evaluating drug combinations. In vivo methods, such as the subrenal capsular assay (Bogden, 1984) and nude mouse tumor culturing (Noso, 1987) offer the obvious benefit of using a system that allows for evaluation of potential host/drug interactions. However, such assays are, by their nature, excessively cumbersome and expensive to use for adequate sampling to fully evaluate either single agents, or combinations of chemotherapeutic drugs (Kratzke and Kramer, 1996).